Background. CXCL12/CXCR4 signaling is essential in cardiac development and repair, however, its contribution to the Aortic valve stenosis (AVS) remains unclear. In this study, we tested the role of endothelial CXCR4 on the development of AVS. Methods. We generated CXCR4 endothelial cell-specific knockout mice (EC CXCR4 KO) by crossing CXCR4 fl/fl mouse with Tie2-Cre mouse to study the role of endothelial cell CXCR4 in AVS. CXCR4 fl/fl mice with no Tie2-Cre gene present were used as control. Echocardiography was used to assess the aortic valve and cardiac function. Heart samples containing the aortic valve were stained using Alizarin Red for detection of calcification. Masson’s Trichrome staining was used for the detection of fibrosis. Apex samples were stained with wheat germ agglutinin to visualize ventricular hypertrophy. Results. Compared with the control group, the deletion of CXCR4 in endothelial cells led to significantly increased aortic valve peak velocity and aortic valve peak pressure gradient, and decreased aortic valve area and ejection fraction. EC CXCR4 KO mice also developed cardiac hypertrophy evidenced by increased left ventricular mass, diastolic and systolic left ventricle posterior wall (LVPW), cardiac myocyte size, and heart weight (HW) to body weight (BW) ratio. Our data also confirmed increased microcalcifications, interstitial fibrosis, and thickened valvular leaflets of the EC CXCR4 KO mice. Conclusions. We found that the deletion of CXCR4 in endothelial cells leads to aortic valve stenosis and left ventricular hypertrophy. This indicates endothelial cell CXCR4 plays a protective role against AVS, and EC CXCR4 KO mice can be used as a novel model for AVS study.

BackgroundCXCL12/CXCR4 signaling is essential in cardiac development and repair, however, its contribution to aortic valve stenosis (AVS) remains unclear. In this study, we tested the role of endothelial CXCR4 on the development of AVS.Materials and methodsWe generated CXCR4 endothelial cell-specific knockout mice (EC CXCR4 KO) by crossing CXCR4fl/fl mice with Tie2-Cre mice to study the role of endothelial cell CXCR4 in AVS. CXCR4fl/fl mice were used as controls. Echocardiography was used to assess the aortic valve and cardiac function. Heart samples containing the aortic valve were stained using Alizarin Red for detection of calcification. Masson’s trichrome staining was used for the detection of fibrosis. The apex of the heart samples was stained with wheat germ agglutinin (WGA) to visualize ventricular hypertrophy.ResultsCompared with the control group, the deletion of CXCR4 in endothelial cells led to significantly increased aortic valve peak velocity and aortic valve peak pressure gradient, with decreased aortic valve area and ejection fraction. EC CXCR4 KO mice also developed cardiac hypertrophy as evidenced by increased diastolic and systolic left ventricle posterior wall thickness (LVPW), cardiac myocyte size, and heart weight (HW) to body weight (BW) ratio. Our data also confirmed increased microcalcifications, interstitial fibrosis, and thickened valvular leaflets of the EC CXCR4 KO mice.ConclusionThe data collected throughout this study suggest the deletion of CXCR4 in endothelial cells is linked to the development of aortic valve stenosis and left ventricular hypertrophy. The statistically significant parameters measured indicate that endothelial cell CXCR4 plays an important role in aortic valve development and function. We have compiled compelling evidence that EC CXCR4 KO mice can be used as a novel model for AVS.

Takotsubo Syndrome (TTS) is an unmet medical need as there is no “standard of care” with an in‐hospital mortality rate equivalent to patients with an acute myocardial infarction. Characteristic of TTS is systolic ballooning of the left ventricular (LV) apex, simultaneous with contraction of the base. The mechanism(s) underlying this disassociation of mechanical activities in the LV is(are) unknown, however, a murine model may provide insights into this clinical conundrum. Previously, we found that mice null for Kv1.5 channels (KO), a model of inadequate coronary metabolic dilation, exhibit TTS (KOTTC) when stressed by transaortic constriction (TAC). Because of the microvascular link to inadequate dilation, we hypothesized that coronary vessels in the base and the apex of the left ventricle have different phenotypes as shown by variations in gene expression. To address this, we dissected small coronary arteries from the LV apex and base of KO control mice (KOC) and following TAC when the mice exhibited profound TTS. Ventricular function (fractional shortening [FS]) was calculated from echocardiographic (Echo) imaging. The Echo measurements revealed FS of 39±3% and 34±6% in LV base and apex, respectively, in KOC (P=NS). In contrast, FS was significantly changed in KOTTC with FS of ‐9±4% in LV apex and 25±11% in the base (P<0.05, with the negative FS representing systolic ballooning). After the Echo measurements, hearts were removed, placed in a chilled dish, and vessels immediately isolated from the LV apex and base (using a dissecting microscope) were placed in Trizol and flash frozen. Total RNA was extracted and used for RNAseq to profile the transcriptomes of small arteries from the apex and base of KOC and KOTTC groups. RNAseq revealed expression of 197 genes significantly changed in the LV apex versus base of KOTTC mice (P<0.05). The 197 genes represented 24 distinct pathways with some of the most significant comprising neuroactive‐receptor ligand interactions, miRs in cancer, and complement and coagulation cascades. The endothelin pathway was significantly upregulated in small arteries of the LV apex compared to base in KOTTC. Moreover, 460 genes were differentially expressed between the apex of KOTTC versus KOC (P<0.05) representing 37 pathways, including miR’s in cancer and cytokine signaling. The changes in miR expression in cancer may also denote changes associated with cell differentiation and apoptosis. These changes support our concept that regional, apex vs base, differences in vascular gene expression may underlie the basis of Takotsubo Syndrome.